Major histocompatibility complex ("MHC") molecules a central role in T cell mediated immune play responses. T lymphocyte ("T cell") antigen receptors ("TCR") recognize endogenously processed fragments of antigens that are presented to T cells in association with major histocompatibility complex ("MHC") class I or class II molecules.
An individual's T cells recognize and are activated by protein antigens only if a fragment of the antigen is properly presented on the surface of a target cell. The antigen presentation process that allows an antigen to be recognized by a T cell requires that the antigen be associated with either (MHC) class I histocompatibility molecules for presentation to cytotoxic T lymphocytes ("CTLs") or class II histocompatibility molecules for presentation to helper T cells. Other T cell subsets such as .gamma./.delta. (gamma-delta) T cells (CD4.sup.--, CD8.sup.--) may recognize alternate "peptide presenting" molecules not encoded in the MHC, such as CDl, etc.
The subset of T cells denoted CD8.sup.+ recognize antigenic determinants/epitopes that are associated with class I histocompatibility molecules. The other subset of T cells, CD4.sup.+ cells, recognize antigenic determinants/epitopes that are associated with class II histocompatibility molecules. The antigenic determinants/ epitopes that are presented on the surface of cells in association with MHC molecules are also known as T cell epitopes.
The study of CD8.sup.+ T cell recognition of target cells has been extensive since the early 1970's when it was demonstrated that CTL recognition of viral-infected autologous target cells requires the presence of self class I MHC molecules. Thus such recognition of target cells by CD8.sup.+ T cells is referred to as being MHC class I-restricted. Zinkernagel, R. M., et al., Adv. Immunol. 27:51 (1979); Doherty, P. C., et al., Adv. Cancer Res. 42:1 (1984); and Zinkernagel, R. M., et al., Nature 248:701 (1974), the disclosures of which are incorporated herein by reference. It was later shown that virus-specificity of CTL's is directed against vital protein-derived peptide sequences that are presented by infected cell MHC class I molecules to CD8.sup.+ T cells. See for example, Townsend, A., et al., Cell 42:457 (1985) and Townsend, A., et al., Cell 44:959 (1986), the disclosures of which are incorporated herein by reference.
Class I molecules (HLA-A, B, C in humans) are composed of two polypeptide chains: a heavy chain which spans the membrane bilayer, and the non-covalently attached light chain, .beta..sub.2 m microglobin (".beta..sub.2 m"). The extracellular portion of the heavy chain is divided into three domains, .alpha..sub.1, .alpha..sub.2, and .alpha..sub.3, each being approximately 90 amino acids. It is not known if free heavy chains inside the cell can bind peptides, or if .beta..sub.2 m must first bind to the heavy chains.
As noted above, it is not the entire antigen that is presented by target cells and recognized by CD8.sup.+ cells, but rather what is presented and recognized are small endogenously processed peptides that are generated from antigens by intracellular degradation pathways in either the cytosol or the endoplasmic reticulum ("ER") of the target cell. Such processed peptides bind to newly synthesized class I heavy chains either before or after binding to .beta..sub.2 m in the secretory compartment. See, for example, Yewdell, J.W., et al., Science 244:1072 (1989); Townsend, A., et al., Cell 62:285 (1990); and Nuchtern, J.G., et al., Nature 339:223 (1989), the disclosures of which are incorporated herein by reference. The processed peptide is bound to the class I heavy chain-light chain dimer molecule via the class I antigen binding site/peptide cleft. The complex thereby generated is a transport competent trimer as reported by Yewdell, J. W., et al., Science 244:1072 (1989); Townsend, A., et al., Cell 62:285 (1990); and Nuchtern, J. G., et al., Nature 339:223 (1989). This class I histocompatibility molecule-processed peptide complex is then transported through the Golgi apparatus, where glycan modification occurs and is expressed on the surface of the target cell where it may be ultimately recognized by T cell clonotypic receptors on CD8.sup.+ cells in conjunction with CD8 accessory molecules. See, Rotzschke, O., et al., Nature 348:252 (1990); Van Bleek, G. M., et al., Nature 348:213 (1990); Rotzschke, O., et al., Science 249:283 (1990); and Falk, K., et al., Nature 348:248 (1990), the disclosures of which are incorporated herein by reference.
Mutant cell lines such as RMA-S, .174, and a somatic cell hybrid of .174 called T2 have been reported in which class I molecules are synthesized but are not stably expressed at the plasma membrane. Se e, for example, Salter, R. D., et al., Immunogenetics 2 1:235 (1985); Salter, R. D., et al., EMBO J. 5:943 (1986), the disclosures of which are incorporated herein by reference. Treatment of such cells with synthetic peptides was found to increase the levels of serologically detectable class I molecules at the cell surface. See, for example, Townsend, A., et al., Nature 240:443 (1989); Cerundolo, V., et al., Nature 345:449 (1990), the disclosures of which are incorporated herein by reference. It was recently shown in murine RMA-S cells that "empty" class I molecules, consisting of heavy chains and .beta..sub.2 m but lacking processed peptide that appeared at the cell surface after growth at 26.degree. C., could bind exogenous peptides more readily than class I molecules on normal RMA-S cells. Ljunggren H. G., et al., Nature 346:476 (1990); and Towsend, A., et al., Cell 62:285, (1990), the disclosures of which are incorporated herein by reference.
Recently, peptides have been isolated from the antigen binding sites of human and murine class I and class II molecules and directly sequenced. Two principal methods have been used to isolate such peptides. In one of the two methods total cellular extraction of such peptides is carried out in pH 2.0 trifluoroacetic acid ("TFA"). This method results in cell cytolysis and release of total cytosolic peptides, only a fraction of which are actually class I-related. This method also typically employs protease inhibitors since cell cytolysis results in the release of proteolytic enzymes that can alter or destroy peptides of potential interest. See, Rotzschke, O., et al., Nature 348:252 (1990), and Falk, K., et al., Nature 348:248 (1990), the disclosures of which are incorporated herein by reference. The second isolation method entails acid denaturation of immunoaffinity purified class I-peptide complexes. By contrast with the first method, the second method of peptide isolation is highly class I selective, and even class I allele specific since monoclonal antibodies directed against individual class I allotypes can be used to immunopurify class I complexes. By this latter approach, the majority of known class I-bound peptide sequence data has been acquired. See, for example, Van Bleek, G. M., et al., Nature 348:213 (1990); Rotzschke, O., et al., Science 249:283 (1990); Madden, D. R., et al., Nature 353:326 (1991); Jardetzky, T. S., et al., Nature 351:290 (1991); and Nikolic-Zugic, J., et al., Immunol. Rev. 10:54 (1991), the disclosures of which are incorporated herein by reference.
The main drawback of these two methods is that since both require cell cytolysis, a large number of starting cells (10.sup.9 -10.sup.11) are required from which peptides are extracted after cellular cytolysis in order to obtain sequence grade quantities (approximately 1 pM) of specific peptide. Therefore the application of such techniques are limited to cell types which readily adapt to in vitro cell culture and which proliferate sufficiently well to allow such high cellular yields.
Methods of isolating class I peptide complexes are of additional interest because CD8+ lymphocytes have emerged as being potentially useful in the development of anti-tumor vaccines, which vaccines will ideally provoke anti-tumor immune responses in individuals. To that end, tumor infiltrating lymphocytes (TILs) have been found to be important agents in the generation of cellular immunity through their identification in spontaneously regressing lesions in some patients as reported by Kornstein, M. J., et al. Cancer Res. 43:2749 (1983), the disclosure of which is incorporated herein by reference. TILs are also frequently found in non-regressing lesions and when present in high numbers are correlated with a better clinical prognosis. Van Duinen, S. G., et al., Cancer Res. 48:1019 (1988), the disclosure of which is incorporated herein by reference. Numerous studies have shown that such TILs display potent anti-melanoma cytolytic activity when they are cultured in vitro with interleukin-2. See, for example, Lotze, M. T., Pigment Cell 10:163 (1990), and Rosenberg, S. A., et al., N. Eng. J. Med. 319:1676 (1988). Anti-melanoma cytolytic activity is typically associated with CD8+ TIL subpopulations which recognize tumor cells in a class I-restricted manner. The HLA class I antigen, HLA-A2, appears to represent the most common class I restriction element for human melanoma TIL, however, other HLA class I antigens such as HLA-A1, -A10, -A24, -A31, -B44, -B50, and -CW7 have also been identified. The identification of such restriction elements may be important in the development of effective melanoma vaccines.
There remains a need for methods that will efficiently extract class I-associated peptides without affinity purification and acid extraction of class I complexes and which requires fewer cells than the aforedescribed methods.